1. Field of the Invention
The present invention refers to an activity based probe (ABP) comprising a glycosidase inhibitor, in particular a beta-glucosidase inhibitor, preferably covalently binding to a glycosidase comprising a detection-group.
2. Description of Related Art
Over the past decade, activity based protein profiling has been used to study many biological processes. The activity based probes (ABPs) used in these studies generally consist of three elements: a warhead, a linker/recognition element and ligation handle or identification tag. The enzyme reacts selectively with the warhead forming a covalent adduct which can then be visualized either by direct visualization (e.g the identification tag is present on the probe) or by two step labeling (e.g post-labeling modification of the ligation handle). This strategy has been widely applied to study proteases such as cathepsins, the proteasome, and esterases such as acetylcholinesterase.
With regard to glycosidases, in particular exo-glycosidases, only a few examples of ABPs are known. Vocadlo and Bertozzi describe a galactosidase probe, which is based on the activated fluorinated glycosides, a class of mechanism based inhibitors of glycosidases. The anomeric center of this galactosidase probe is activated by protonation by one of two carboxylic acids present in the active site of the glycosidase. The other carboxylic acid attacks the anomeric center forming a glycosyl-enzyme complex. The fluorine residue in the galactosidase probe slows formation as well as hydrolysis of the glycosyl-enzyme complex down. The activated leaving group increases the reaction rate of the first step leading to accumulation of the inhibitor complex. Although the complex is stabilized, the acylal ester linkage slowly hydrolyzes due to the endo-cyclic oxygen. Lifetimes ranging from seconds to months have been reported for these complexes. Despite this disadvantage, all ABPs reported so far are based on this class of mechanism-based inhibitors.
Numerous lysosomal storage disorders are based on malfunctions of one or more enzymes, for example glucosidases, involved in the metabolism of different substances. A storage disorder is any disease or condition which is characterised by the abnormal accumulation and storage of material within the cells. The stored material will vary depending upon the type of condition. One of the most common lysosomal storage disorders is Gaucher disease, which is characterized by a defective catabolism of glucosylceramide based on the malfunctioning of the enzyme glucocerebrosidase (GBA1). GBA1 is an acid hydrolase that primarily degrades its substrate in acidic compartments such as endosomes and lysosomes, particularly of macrophages. Increasing GBA1 activity is the rational target for therapy of Gaucher disease. This can be accomplished by enzyme therapy, by which mannose-terminated GBA1 upon intravenous infusion reaches lysosomes of macrophages Clinical response to enzyme therapy is spectacular, but their remain disadvantages. Firstly, the costs are extremely high (>100.000ε/adult patient/year) and secondly, enzyme therapy does not prevent neurological manifestations given the inability of enzymes to effectively cross the blood-brain barrier.
Hence, since many years intensive research is ongoing to identify a therapeutic for effective treatment of storage disorders such as Gaucher disease. So-called substrate reduction therapy, using iminosugars or ceramide-mimics, aims to reduce biosynthesis of glucosylceramide in Gaucher patients, thus restoring the balance with impaired degradation. The common drugs for treating Gaucher disease these days (for example Zavesca, GENZ) show severe side-effects leading to gastrointestinal complications, such as flatulence and severe diarrhea resulting in abrupt weight loss, and poorly penetrate the brain. An interesting new approach for treating storage disease is the use of chaperones. The concept of chaperones implies that particular (point) mutant forms of an enzyme such as GBA1 can be assisted in their folding and stability by chaperones interacting with the catalytic pocket of the enzyme. For example isofagomine and hydrophobic iminosugars are considered as promising chaperones. When cells containing mutant GBA1 are incubated for some days with such chaperones (at concentrations close to IC50 values), indeed an increased GBA1 activity is measured in cell lysates and increased GBA1 protein is detected by immunofluorescence. Unfortunately, it has not yet been documented that endo/lysosomal degradation of the glucosidase's substrate such as glucosylceramide improves, i.e., increases by chaperone treatment.
The lack of experimental evidence results from the fact that measurement of an increase in active enzyme in situ is difficult. No reliable methods for its measuring exists. Presently, some groups test the effect of chaperone treatment by incubating cells for 1 hour in acetate buffer (pH 4.0) containing the artificial substrate 4-methylumbelliferyl-beta-D-glucoside. It is far from being clear that under this condition the cellular surrounding of GBA1 is not fundamentally changed. Other research groups have measured GBA1 activity in living cells using fluoresceine-diglucoside (FDG) or fluorescent NBD-glucosylceramide as substrates. FDG is cleaved by GBA1 to generate fluorescent fluoresceine that can be detected for example by FACS. Fluorescent C6-NBD-GlcCer is added to cells, where after it is intracellularly converted to C6-NBD-Cer by GBA1. After harvesting the cells and extraction of lipids, the fluorescent NBD-glucosylceramide and NBD-ceramide can be separated, for example by thin layer chromatography, and be quantified. These advanced assays require considerable expertise, are labour-intensive and cumbersome.
Hence, it is presently problematic to assess the value of small compounds as effective chaperones for stabilization of a defect glucosidase such as GBA1 and thus, their therapeutic value for the treatment of lysosomal storage disorders such as Gaucher disease. Researchers presently still rely on artificial assays that poorly reflect a chaperone-mediated increase in in vivo degradative capacity, see for example Mu T W, Ong D S, Wang Y J, Balch W E, Yates J R 3rd, Segatori L, Kelly J W, Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell 2008 Sep. 5; 134(5):769-81.
The present invention now provides the development of highly efficient ABPs interacting with a glycosidase, providing basis for diagnosing a storage disorder and/or screening of compounds for preventing and/or treating a storage disorder such as Gaucher disease.
The present invention is further directed to efficient ABPs interacting with a glycosidase, providing basis for in vitro or in vivo monitoring of glycosidases in living organisms and cells.
Moreover, the present invention refers to efficient ABPs interacting with a therapeutic glycosidase, providing basis for in vitro or in vivo monitoring of a pre-labeled enzyme with respect to its delivery to lysosomes in cells and tissues of model animals and patients.
Further, the present invention relates to efficient ABPs interacting with a glycosidase-fusion protein, providing basis for ultra-sensitive in vitro or in vivo monitoring of the fusion protein.